CN109091490A - Adenosine and autophagy inhibitor combination are preparing the application in treatment of colon cancer drug - Google Patents
Adenosine and autophagy inhibitor combination are preparing the application in treatment of colon cancer drug Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
- A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
- A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4706—4-Aminoquinolines; 8-Aminoquinolines, e.g. chloroquine, primaquine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
- A61K31/52—Purines, e.g. adenine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
- A61K31/585—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin containing lactone rings, e.g. oxandrolone, bufalin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
The present invention relates to technical field of pharmaceuticals, specifically adenosine and autophagy inhibitor combination is preparing the application in treatment of colon cancer drug.The invention has the advantages that: present invention firstly discovers that adenosine is in colon cancer cell by inhibiting PI3K/Akt/mTOR signal path to induce autophagy；From phenotype occur sequentially, adenosine induction apoptosis it is first, autophagy occur rear.The ratio for the Apoptosis of Colon Cancer Cells that the expression for lowering autophagy related gene can promote adenosine to induce, therefore adenosine joint autophagy inhibitor (such as chloroquine or 3-MA) can more thoroughly kill colon cancer cell, obtain better clinical therapeutic efficacy.
Description
Technical field
The present invention relates to technical field of pharmaceuticals, specifically, being a kind of biological metabolism ATP derivative: adenosine and autophagy
Inhibitor combination is preparing the application in treatment of colon cancer drug.
Background technique
Colon cancer is the common malignant tumor of digestive tract for betiding colon site.Disease incidence accounts for the 3rd of gastroenteric tumor
Position.Annual whole world new life case is more than 1,000,000, death about 500,000.Morbidity main cause be high-fat recipe and
Fiber insufficiency of intake.Colon chronic inflammation keeps the incidence of intestinal cancer higher than general normal population.The person that has polyp of colon, colon cancer hair
Sick rate is 5 times of no polyp of colon person.Familial multiple intestinal polyp tumor, the incidence of canceration are higher.Inherent cause may
Participate in the morbidity of colon cancer.Although operation excision is effective therapeutic scheme, postoperative recurrence and shifting risk property are still suitable
Ground is high.Postoperative recurrence or is not cut off lesion completely since internal residual cancer cells do mischief.Before laparotomy ventrotomy, usually
Bowel lavage is administered before first carrying out tumour enteric cavity chemotherapy or rectal cancer, cancer cell can be prevented to spread, kill and eliminate cancer cell.Art
After continue chemotherapeutic treatment, it is possible to 5 years survival rates after improving Operation of Colon Carcinoma.For surgical procedure not or it is postoperative be anti-
The patient that rotation stop is moved only is further continued for carrying out chemotherapy, and effect is generally not satisfactory in fact.Just there is an urgent need to us to seek conscientiously for this
Feasible new drug treats this persistent ailment.
Adenosine, purine nucleosides are one of the metabolins of life power ATP, participate in many basic biological processes, including
The biosynthesis of nucleosides and the energetic supersession of cell.It is widely present in tissue, organ and each cell.Adenosine, because
The degradation of ATP and generate, and be released to extracellular.Intracellular, adenosine can also further be metabolised to inosine and time yellow fast
Purine.In recent years, have and more and more report that effects of adenosine on cellular growth and differentiation have a major impact.Extracellular adenosine is generally through two
Different mechanisms are planted to influence cell.One is foreign aid's mechanism.The adenosine receptor of adenosine and cell membrane surface be combined with each other, these by
Body raises all kinds of G-proteins intracellular again influences target cell to activate respective signal path.The other is by inherent logical
Road plays biological activity.The adenosine transport body of cell membrane surface is directly had an effect with adenosine, adenosine is taken in intracellular.Gland
Glycosides is finally converted to AMP by adenosine kinase in the cell, then influences the activity of AMP-activated Protein Kinase AMPK.
Up to the present, have more literatures report adenosines can in kinds of tumor cells inducing apoptosis ([1]
D.Yang,T.Yaguchi,C.-R.Lim,Y.Ishizawa,T.Nakano,T.Nishizaki,Tuning of
apoptosis-mediator gene transcription in HepG2 human hepatoma cells through
an adenosine signal,Cancer Letters 291(2010)225-229.[2]S.G.Kim,G.Ravi,
C.Hoffmann,Y.J.Jung,M.Kim,A.S.Chen,K.A.Jacobson,p53-independent induction of
Fas and apoptosis in leukemic cells by an adenosine derivative,Cl-IB-MECA,
Biochemical Pharmacology 63(2002)871-880.[3]P.Mlejnek,P.Dolezel,P.Kosztyu,P-
glycoprotein mediates resistance to A3 adenosine receptor agonist 2-chloro-N-
6-(3-iodobenzyl)-adenosine-5'-n-methyluronamide in human leukemia cells,
Journal of Cellular Physiology 227(2012)676-685.[4]S.Cohen,S.M.Stemmer,
G.Zozulya,A.Ochaion,R.Patoka,F.Barer,S.Bar-Yehuda,L.Rath-Wolfson,
K.A.Jacobson,P.Fishman,CF102 an A(3)Adenosine Receptor Agonist Mediates Anti-
Tumor and Anti-Inflammatory Effects in the Liver,Journal of Cellular
Physiology 226(2011)2438-2447.[5]S.Morello,R.Sorrentino,A.Porta,G.Forte,
A.Popolo,A.Petrella,A.Pinto,CI-IB-MECA Enhances TRAIL-Induced Apoptosis via
the Modulation of NF-kappa B Signalling Pathway in Thyroid Cancer Cells,
Journal of Cellular Physiology 221(2009)378-386.[6]Y.Yasuda,M.Saito,
T.Yamamura,T.Yaguchi,T.Nishizaki,Extracellular adenosine induces apoptosis in
Caco-2 human colonic cancer cells by activating caspase-9/-3via A(2a)
adenosine receptors,Journal of Gastroenterology 44(2009)56-65.[7]K.Tamura,
T.Kanno,Y.Fujita,A.Gotoh,T.Nakano,T.Nishizaki,A2a adenosine receptor mediates
HepG2 cell apoptosis by downregulating Bcl-XL expression and upregulating bid
expression,Journal of Cellular Biochemistry 113(2012)1766-1775.[8]J.S.Long,
D.Crighton,J.O'Prey,G.MacKay,L.Zheng,T.M.Palmer,E.Gottlieb,K.M.Ryan,
Extracellular Adenosine Sensing-A Metabolic Cell Death Priming Mechanism
Downstream of p53,Molecular Cell 50(2013)394-406.[9]T.-i.Otsuki,T.Kanno,
Y.Fujita,C.Tabata,K.Fukuoka,T.Nakano,A.Gotoh,T.Nishizaki,A(3)Adenosine
Receptor-Mediated p53-Dependent Apoptosis in Lu-65Human Lung Cancer Cells,
Cellular Physiology and Biochemistry 30(2012)210-220.[10]M.Aghaei,F.Karami-
Tehrani,M.Panjehpour,S.Salami,F.Fallahian,Adenosine induces cell-cycle arrest
and apoptosis in androgen-dependent and-independent prostate cancer cell
lines,LNcap-FGC-10,DU-145,and PC3,Prostate72(2012)361-375.[11]M.Aghaei,
M.Panjehpour,F.Karami-Tehrani,S.Salami,Molecular mechanisms of A3 adenosine
receptor-induced G1 cell cycle arrest and apoptosis in androgen-dependent and
independent prostate cancer cell lines:involvement of intrinsic pathway,
Journal of Cancer Research and Clinical Oncology 137(2011)1511-1523.[12]
L.Madi,S.Bar-Yehuda,F.Barer,E.Ardon,A.Ochaion,P.Fishman,A3 adenosine receptor
activation in melanoma cells-Association between receptor fate and tumor
growth inhibition,Journal of Biological Chemistry 278(2003)42121-42130.[13]
P.Fishman,S.Bar-Yehuda,G.Ohana,F.Barer,A.Ochaion,A.Erlanger,L.Madi,An agonist
to the A(3)adenosine receptor inhibits colon carcinoma growth in mice via
modulation of GSK-3 beta and NF-kappa B,Oncogene 23(2004)2465-2471.[14]
P.Fishman,L.Madi,S.Bar-Yehuda,F.Barer,L.Del Valle,K.Khalili,Evidence for
involvement of Wnt signaling pathway in IB-MECA mediated suppression of
melanoma cells,Oncogene 21(2002)4060-4064.[15]J.Lu,A.Pierron,K.Ravid,An
adenosine analogue,IB-MECA,down-regulates estrogen receptor a and suppresses
human breast cancer cell proliferation,Cancer Research 63(2003)6413-6423.[16]
H.Chung,J.Y.Jung,S.D.Cho,K.A.Hong,H.J.Kim,D.H.Shin,H.Kim,H.O.Kim,D.H.Shin,
H.W.Lee,L.S.Jeong,G.Kong,The antitumor effect of LJ-529,a novel agonist to A3
adenosine receptor,in both estrogen receptor-positive and estrogen receptor-
negative human breast cancers,Molecular Cancer Therapeutics 5(2006)685-692.
[17]S.Morello,A.Petrella,M.Festa,A.Popolo,M.Monaco,E.Vuttariello,
G.Chiappetta,L.Parente,A.Pinto,Cl-IB-MECA inhibits human thyroid cancer cell
proliferation independently of A3 adenosine receptor activation,Cancer
Biology&Therapy 7(2008)278-284.[18]D.Yang,T.Yaguchi,H.Yamamoto,T.Nishizaki,
Intracellularly transported adenosine induces apoptosis in HuH-7 human
hepatoma cells by downregulating c-FLIP expression causing caspase-3/-8
activation,Biochemical Pharmacology 73(2007)1665-1675.[19]D.Yang,T.Yaguchi,
T.Nakano,T.Nishizaki,Adenosine-induced caspase-3 activation by tuning Bcl-X-
L/DIABLO/IAP expression in HuH-7 human hepatoma cells,Cell Biology and
Toxicology 26(2010)319-330.[20]Y.Nogi,T.Kanno,T.Nakano,Y.Fujita,C.Tabata,
K.Fukuoka,A.Gotoh,T.Nishizaki,AMP Converted from Intracellularly Transported
Adenosine Upregulates p53 Expression to Induce Malignant Pleural Mesothelioma
Cell Apoptosis,Cellular Physiology and Biochemistry 30(2012)61-74.[21]D.Yang,
T.Yaguchi,T.Nakano,T.Nishizaki,Adenosine Activates AMPK to Phosphorylate Bcl-
X-L Responsible for Mitochondrial Damage and DIABLO Release in HuH-7 Cells,
Cellular Physiology and Biochemistry 27(2011)71-78.[22]K.Sai,D.Yang,
H.Yamamoto,H.Fujikawa,S.Yamamoto,T.Nagata,M.Saito,T.Yamamura,T.Nishizaki,A(1)
adenosine receptor signal and AMPK involving caspase-9/-3activation are
responsible for adenosine-induced RCR-1astrocytoma cell death,
Neurotoxicology27(2006)458-467.[23]C.Cande,F.Cecconi,P.Dessen,G.Kroemer,
Apoptosis-inducing factor(AIF):key to the conserved caspase-independent
Pathways of cell death?, 115 (2002) 4727-4734. [24] of Journal of Cell Science
T.Kanno,A.Gotoh,Y.Fujita,T.Nakano,T.Nishizaki,A(3)Adenosine Receptor Mediates
Apoptosis in 5637Human Bladder Cancer Cells by G(q)Protein/PKC-Dependent AIF
Upregulation, Cellular Physiology and Biochemistry30 (2012) 1159-1168.), however, extremely
In adenosine induced in human colon cancer cell autophagy there is no literature reported on.Autophagy is an extremely complex signals-modulating access,
It is that eukaryocyte is made and stress be coped in real time to thousand ten thousand unreal external environments of change continuously and healthily.The knot that autophagy is induced in adenosine
It plays a role in colon-cancer cell apoptosis and corresponding molecular mechanism is still indefinite.
Summary of the invention
It is an object of the invention to be directed to colon cancer drug unsatisfactory curative effect, postoperative recurrence and shifting risk property are still relatively high
This status provides a kind of efficient scheme for colorectal cancer clinical treatment.It is another object of the invention to provide a kind of colons
The potential target of cancer treatment, to inhibit the proliferation of remaining colon cancer cell.
To achieve the above object, the technical solution adopted by the present invention is that:
The first aspect of the present invention, provides adenosine and autophagy inhibitor combination is preparing the application in treatment of colon cancer drug
Further, the autophagy inhibitor promotes Apoptosis of Colon Cancer Cells caused by adenosine.
Further, the autophagy inhibitor is inhibition/downward autophagy related gene expression substance.
Further, the autophagy related gene is Beclin1 or ATG5.
Further, the autophagy inhibitor is the protein expression for inhibiting autophagy related gene Beclin1 or ATG5
Substance.
Preferably, the autophagy inhibitor is chloroquine (CQ) or 3-MA (3-MA).
Preferably, the autophagy inhibitor can also be BafilomycinA1 (Bafilomycin A1),
Hydroxychloroquine (hydroxychloroquine), Wortmannin (wortmannin).
The second aspect of the present invention, provides a kind of pharmaceutical composition for treating colon cancer, and the pharmaceutical composition includes
Adenosine and autophagy inhibitor and other pharmaceutically acceptable auxiliary material or carriers.
The third aspect of the present invention provides autophagy inhibitor in preparing Apoptosis of Colon Cancer Cells promotor caused by adenosine
Application.
Further, the autophagy inhibitor is inhibition/downward autophagy related gene expression substance.
Further, the autophagy related gene is Beclin1 or ATG5.
Further, the autophagy inhibitor is the protein expression for inhibiting autophagy related gene Beclin1 or ATG5
Substance.
Preferably, the autophagy inhibitor be chloroquine (CQ), 3-MA (3-MA), BafilomycinA1 (bar
Buddhist Lip river mycin A1), Hydroxychloroquine (hydroxychloroquine) or Wortmannin (wortmannin).
The invention has the advantages that:
Present invention firstly discovers that adenosine is in colon cancer cell by inhibiting PI3K/Akt/mTOR signal path to induce autophagy
Occur；From phenotype occur sequentially, adenosine induction apoptosis it is first, autophagy occur rear.Lower autophagy related gene
The ratio for the Apoptosis of Colon Cancer Cells that expression can promote adenosine to induce, therefore adenosine combines autophagy inhibitor (such as chloroquine or 3- first
Base adenine) colon cancer cell can be more thoroughly killed, obtain better clinical therapeutic efficacy.
Detailed description of the invention
Fig. 1: adenosine induces colon cancer cell that autophagy figure occurs；A-b: after adenosine processing, LC3-II isomers and autophagy phase
The increase of concentration gradient and time gradient is presented to adenosine drug for the gene beclin 1 of pass and the expression quantity of ATG5.(a:
SW620 and b:SW480).At aobvious in the colon cancer cell that the ratio of the dotted positive cell of c-d:GFP surely turns EGFP-LC3 at two plants
It writes and increases (c:SW620-EGFP-LC3 and d:SW480-EGFP-LC3).E-f: Acridine orange experiment also confirms that adenosine in colon
Autophagy is induced in cancer cell.(e:SW620 and f:SW480).
Fig. 2: adenosine is by inhibiting PI3K/Akt/mTOR signal path figure.
Fig. 3: active oxygen ROS is to induce the main reason for autophagy occurs for colon cancer cell；A: adenosine induced activity oxygen ROS's
Generation is to rely on external adenosine concentration.B-c: pre- using active oxygen killer nitrogen acetylcysteine NAC or glutathione GSH
Add adenosine drug after processing again, the expression quantity of autophagy marker LC3-II is repressed while finding that apoptosis marker c-PARP is aobvious
It writes and increases (b:SW620, c:SW480), d-e: streaming further detects discovery ROS inhibitor and inhibits the generation of cell autophagy simultaneously
Apoptotic cell ratio is caused to significantly rise (d:SW620, e:SW480).
Fig. 4: the decline of autophagy level of activity, Level of Apoptosis significantly rise.A-d:p62 or ATG5 are inhibited by endogenous
Afterwards, the expression quantity of LC3-II substantially reduces, and the expression quantity of apoptosis marker c-PARP dramatically increases (a, c:SW620；B, d:
SW480).E-h: flow cytometer detection p62 or ATG5 lower after with adenosine coprocessing to Level of Apoptosis influence (e-f:
Sip62, g-h:siATG5, e, g:SW620, f, h:SW480).
Fig. 5: the order of occurrence of verifying autophagy and apoptosis.A-b:Hoechst33258 and acridine orange AO contaminates altogether confirms that autophagy exists
(a:SW620, b:SW480) occurs before Apoptosis.C-d:Western blot detects intracellular apoptosis-related protein (c-
PARP, c-caspase3) and autophagy GAP-associated protein GAP (p-mTOR, LC3) (c:SW620, d:SW480).
Fig. 6: autophagy inhibitor chloroquine (CQ) or 3-MA (3-MA) and adenosine drug combination processing colon cancer are thin
Born of the same parents' effect.A-b:Western blot has detected adenosine and autophagy inhibitor coprocessing to cell death related protein (c-PARP)
With the influence of autophagy GAP-associated protein GAP (LC3-I/II) expression.C-f: after adenosine and autophagy inhibitor drug, with list added with adenosine
Cell is compared, and cell survival rate is remarkably decreased.(c-e:SW620, d, f:SW480).
Fig. 7: autophagy inhibitor dramatically increases the ratio of the apoptosis-induced cell of adenosine.A, b: autophagy inhibitor CQ (chloroquine),
C, d: autophagy inhibitor 3-MA (3- methyl purine).
Specific embodiment
It elaborates below with reference to embodiment to specific embodiment provided by the invention.
Embodiment 1
One, experimental method
(1) cell culture and reagent
Human colon cancer cell SW620 and SW480 are purchased from Chinese Academy of Sciences's cell bank.Cell culture is in DMEM+10%FBS.CQ,3-
MA, Hoechst33258 and acridine orange AO are purchased from Sigma company.2000 are purchased from Invitrogen public affairs
Department.Main antibody ATG5, Beclin1, p-Akt, mTOR, p-mTOR, p-70S6K, p-EBP1, p62 are purchased from Cell
Signaling Inc, Danvers, USA, LC3 are purchased from Abclonal, USA, and Actin is purchased from health vitamins company.
(2) recovery of colon carcinoma cell line and secondary culture
Cell recovery: SW620 cell and SW80 cell are taken out from -80 DEG C of ultra low temperature freezers, are put into 37 DEG C of water-baths
In, melt completely to it, is transferred in the 15ml centrifuge tube with 5ml culture medium, 1200rpm is centrifuged 5min.Supernatant is abandoned,
The fresh DMEM culture medium containing 10%FBS of 1ml is added, after mixing, is transferred to the 10cm culture dish with 8ml culture medium
In, it is placed in after mixing in 37 DEG C of cell incubators and carries out secondary culture；
Cell changes liquid: cell culture medium is sucked out, 1 × PBS is added and cleans 2 times suctions, it is big according to culture dish/cell plates
The small fresh culture that respective volume is added；
Cell passage: can be passed on when the cell density in culture dish reaches 90% or more, and culture medium is sucked out, adds
1 × PBS cleans 2 times suctions, adds 500 μ l Trypsin-EDTA (10cm culture dish).To cell dissociation at free individual cells
When, culture medium is added and terminates digestion, piping and druming mixes, and passes three ratios according to one, is passed in new culture dish, and culture medium is added and is
It can；
Cell cryopreservation: DMEM culture medium of the configuration containing 10%DMSO is placed on 4 DEG C of refrigerator pre-coolings；By cell dissociation at
After free cell, culture medium is added and terminates digestion, and liquid is transferred to 15ml centrifuge tube.
(3) cell transfecting and overexpression
RNAiMax transfects siRNA, 2000 transfected plasmids of Lipofectamine.Sip62 and siATG5 interference sequence is purchased from
U.S. lucky biological Co., Ltd；PcDNA3 and pcDNA3-GFP-LC3.
(4) CCK8 is tested
Cell is sowed into 96 orifice plates.SW620 cell is sowed 2500/hole, and SW80 cell is sowed 1600/hole.Overnight
Incubator is incubated for, adherent to cell.Agent-feeding treatment was carried out to cell in second day.Before needing to detect, training liquid is discarded, CCK8 is molten
Liquid is diluted in 1:10 ratio with complete medium, and every 100 μ l of hole is placed in cell incubator 3-4 hours.Utilize microplate reader
SPECTRAmax PLUS38 detects absorption peak of the solution at 50nm.
(5) apoptosis detection (Annexin V, PI)
1. collecting cell: the cell after 1 × PBS is rinsed is digested with 0.25% pancreatin in 37 DEG C of insulating boxs.To cell
When being digested to free individual cells, culture medium is added and terminates digestion, piping and druming mixes.1500rpm, 5min, 4 DEG C of centrifugations, discard
Supernatant.1500rpm, 5min, 4 DEG C of centrifugations again, discard supernatant；
2. being resuspended: the 1 × Annexin V Binding Solution prepared in advance is added, is made final concentration of 1 × 106
The cell suspension of a cell/ml；
3. dyeing: 3 μ l Annexin V, FITC conjugates being added into cell suspension, add the PI solution of 3 μ l, room
Temperature is protected from light upper machine testing after incubation 15min.
Whole process is placed in be operated on ice.
(6) total protein of cell is extracted
1. collecting cell: the cell after 1 × PBS is rinsed is digested with 0.25% pancreatin in 37 DEG C of insulating boxs.To cell
When being digested to free individual cells, culture medium is added and terminates digestion, piping and druming mixes.1500rpm, 5min, 4 DEG C of centrifugations, discard
Supernatant.1500rpm, 5min, 4 DEG C of centrifugations again, discard supernatant；
2. cracking: imitating lysate in addition, blow even.30min is cracked, is vortexed and shakes every 10min；
3. centrifugation: supernatant is collected in 12500rpm, 10min, 4 DEG C of centrifugations.
Whole process is placed in be operated on ice.
(7) BCA protein quantification
1. the formulation of standard protein concentration curve: taking the EP of 5 1.5ml to manage, be separately added into 25,5,30 μ l BSA (2mg/
Ml then the deionized water of 0,15,30 μ l is respectively added again, is sufficiently mixed by inversion for) standard protein, constitute A, B and C tri- and manage；Again from
B, 20 μ l liquid of C pipe taking-up, which are sequentially added to D and E, manages, and adds 20 μ l deionized waters in D and E pipe, is sufficiently mixed by inversion.Point
Do not take respectively 25 μ l that 96 orifice plates are added from five pipes, 200 μ l BCA working solutions are added in every hole, mix；37 DEG C of insulating boxs are incubated for 30min
Afterwards, light absorption value is detected at 562nm with microplate reader.
2. the measurement of laboratory sample concentration: taking 5 μ l samples to be added in 96 orifice plates, 20 μ l deionizations are added in sample well
Water mixes.200 μ l BCA working solutions are added in every hole, mix；After 37 DEG C of insulating boxs are incubated for 30min, with microplate reader at 562nm
Detect light absorption value.Finally sample concentration is carried out according to the OD value of the standard protein sample of measurement and referring to the concentration curve of albumin
Calculating；
(8) Western Blot
1. the preparation of protein sample: albumen needed for being calculated according to the protein concentration of measurement and according to required albumen applied sample amount
Sample volume；Prepare the protein sample that total volume is 20 μ l；After 95 DEG C of denaturation 5min, -20 DEG C save or directly carry out SDS-
PAGE gel electrophoresis.
2.SDS-PAGE electrophoresis: electrophoresis is carried out after protein sample is added to PAGE gel；80V first, 30min；
After bromine meal with wine indigo plant fully enters separation gel, constant pressure 120V, until bromine drinks blue run to the edge of gel；
3. transferring film: methanol activates pvdf membrane, carries out transferring film, constant current 170mA, 2hr after 1min；
4. closing: closing 2hr containing 5% skimmed milk power；
5. primary antibody is incubated for: after the completion of closing, 1 × TBST is rinsed 3 times, every all over 5min；Primary antibody, 4 DEG C of overnight incubations are added；
6. secondary antibody is incubated for: primary antibody is rinsed 3 times after the completion of being incubated for 1 × TBST, every all over 5min；Secondary antibody, incubation at room temperature is added
2hr；
7. development: secondary antibody is rinsed 3 times after the completion of being incubated for 1 × TBST, every all over 5min；It is super quick that ECL is added after the completion of cleaning
Luminous substrate is developed after pvdf membrane is sufficiently reacted with substrate.
(9) tolerance of the cell to G418
1. G418 is diluted to 200 μ g/ml, 400 μ g/ml, 600 μ g/ml, 800 μ g/ml and 1000 μ with complete medium
g/ml；
2.6 orifice plates spread cell, ten thousand cells of every hole 10-20；Liquid is changed after 12 hours, changes the screening training of G418 concentration gradient into
Support base；Screening and culturing medium is changed every other day.On the basis of the G418 concentration for cultivating 90% cell death in 5-7 days, as primary dcreening operation concentration.
(10) foundation of stable cell strain
1. dividing disk into 6 orifice plates after plasmid transfection 2 hours, ten thousand cells of every hole 10-15；
2. second day after point disk, abandoning training liquid, 1 × PBS is rinsed one time, changes the screening and culturing medium of G418 concentration gradient into, often
Liquid was changed every three days；
3. visible resistance clone after screening 1 day.
(11) airflow classification
1. collecting cell: the cell after 1 × PBS is rinsed is digested with 0.25% pancreatin in 37 DEG C of insulating boxs.To cell
When being digested to free individual cells, culture medium is added and terminates digestion, piping and druming mixes.1500rpm, 5min, 4 DEG C of centrifugations, discard
Supernatant；
2. cell is transferred in 15ml centrifuge tube, and in the dual anti-mycillin mixed liquor for being wherein added 50%；
3. machine screening purifying on, screens ratio: choosing preceding 5%；
4. cell after purification is added 6cm culture dish, 50% dual anti-and corresponding G418 solution is added, trained in cell
Support case culture.
(12) acridine orange AO dyeing
1. matching system dye: complete medium is added in 1:2000 ratio in AO
2. discarding culture medium, the dyestuff of appropriate volume is added in the cell after 1 × PBS is rinsed, is incubated in cell incubator
15min；
3. fluorescence microscopy is under the microscope；
(13) Hoechst33258 contaminates altogether with acridine orange AO
1. matching system dye: complete medium is added in 1:2000 ratio in AO
2. discarding culture medium, the dyestuff of appropriate volume is added in the cell after 1 × PBS is rinsed, is incubated in cell incubator
15min；
3. discarding the culture medium containing AO dyestuff, 1 × PBS is changed into, and Hoechst33258, which is added, makes its final concentration be
10ug/ml is incubated for 15min in cell incubator；
4. fluorescence microscopy is under the microscope；
(14) statistical analysis
Each experimental data takes the result of independent experiment three times to carry out two-sided unpaired Student's t inspection
It tests.As a result it is indicated in the form of average value ± SEM, * represents P < 0.05；* represents P < 0.01；* * represents P < 0.001；Ns, generation
Table is without significant difference.
Two, experimental result:
(1) autophagy occurs for adenosine induction colon cancer cell；
After adenosine handles colon cancer cell line, the expression quantity of LC3-II isomers is in two plants of cells of SW620 and SW80
The increase of concentration gradient and time gradient is presented to adenosine drug.The albumen of relevant to autophagy gene beclin 1 and ATG5
Expression quantity also increases with the increase of adenosine concentration for the treatment of and processing time.Concentration ladder is presented to adenosine in the generation of this prompt autophagy
The dependence of degree and time gradient (see Fig. 1 a and b).
Building successfully surely turns GFP-LC3 colon cancer cell line after adenosine is added and handles 72 hours, colon cancer cell
In SW620-GFP-LC3 and SW480-GFP-LC3, by normally spreading in entire cytoplasm pond in the green fluorescence LC3- dispersed
I is converted to a large amount of autophagy LC3-II isomers dots structure.The ratio of the dotted positive cell of GFP is in SW620-EGFP-LC3
Increase to 60.7 ± 2.2% from 2.1 ± 1.9% with the increase of adenosine concentration in cell；This ratio is in SW480-EGFP-
It uprushes from 4.3 ± 2.9% to 54.6 ± 4.8% (P < 0.001 * * *) (see Fig. 1 c and d) in LC3 cell.
Acridine orange experiment also confirms that adenosine induces autophagy in colon cancer cell.Cell is intracellular to there is a large amount of red
Fluorescence vesicle, and non-dosing group then rarer red fluorescence vesicle.Statistics discovery handles 72 hours SW620 in 3mM adenosine
In, red fluorescence point quantity increases to 76.2 ± 2.5% from 1.5 ± 0.%；In the SW480 of 1mM adenosine processing, this ratio
Then surge from 2.1 ± 0.8% to 89 ± 2.1%.Data result has statistical significance (P < 0.001 * * *) (see Fig. 1 e and f).
(2) adenosine is by inhibiting PI3K/Akt/mTOR signal path to induce autophagy；
For the signal path relevant to autophagy of clear adenosine induction, Western blot confirms thin in SW620 and SW480
In born of the same parents, the processing of adenosine causes the dephosphorylation of AKT and the inactivation of mTOR.MTOR is in entire protein level and phosphorylation level
All it is remarkably decreased.The inactivation of mTOR reduces the phosphorylation level of its substrate 4EBP1 and 70S6K significantly.This confirms mTOR letter
To handle induced autophagy closely related (see Fig. 2) for adenosine in colon cancer cell for number access.
(3) autophagy induced factor；
Previous experiments confirm that adenosine can be with the generation of induced activity oxygen ROS (see Fig. 3 a).Utilize active oxygen killer nitrogen second
Add adenosine drug again after acyl cysteine NAC or glutathione GSH pretreatment colon cancer cell, autophagy marker LC3-II's
Expression quantity is suppressed, and c-PARP expression quantity increases (see Fig. 3 b and c), and the spy of the apoptosis such as round shrinkage is also presented in the form of cell
Sign, apoptotic cell ratio significantly rise (see Fig. 3 d and e).This prompt active oxygen ROS is to induce colon cancer cell autophagy occurs
Main cause.
(4) relationship between the autophagy and apoptosis of adenosine induction；
The expression of autophagy related gene p62 or ATG5 are lowered using RNA perturbation technique, and cell is handled to observe adenosine with this
Variation.Western Blot verifies p62 or ATG5 interference effect, and detects autophagy GAP-associated protein GAP (LC3-I/ with corresponding antibodies
) and the expression of apoptosis-related protein (c-PARP) II.After p62 or ATG5 is inhibited by endogenous, the expression quantity of LC3-II is aobvious
It writes and reduces, and the expression quantity of caspase3 substrate c-PARP dramatically increases.This explanation, the decline of autophagy level of activity, Apoptosis
Level significantly rises (see Fig. 4 a b c and d).
It is detected using the method for apoptosis detection after p62 or ATG5 is lowered with adenosine coprocessing to Level of Apoptosis
It influences.The results show that 3mM adenosine single treatment 72 hours, SW620 Apoptosis ratio is 21.9 ± 1.%；And p62 or
For ATG5siRNA with after adenosine coprocessing, this ratio rises to 38.0 ± 0.5% and 31.7 ± 0.5%.In SW480 also
To similar situation.P62 or ATG5siRNA and 1mM adenosine coprocessing be after 72 hours, apoptosis ratio from adenosine single treatment 51.0
± 0.% rises to 66.0 ± 0.3% or 60.0 ± 0.2%.The autophagy for confirming adenosine induction is that have protective, protection
Colon cancer is from further apoptosis (see Fig. 4 e f g and h).
In order to verify the order of occurrence of autophagy and apoptosis, Hoechst33258 and acridine orange AO contaminate altogether confirms that autophagy is from 48
Hour starts, and as a child reaches peak in adenosine processing 72；And apoptosis has been then obvious generation at 48 hours (see Fig. 5 a and b).
Western blot detect intracellular apoptosis-related protein (c-PARP, c-caspase3) and autophagy GAP-associated protein GAP (p-mTOR,
LC3) expression in time also confirms that, c-PARP was expressed at 48 hours in SW620 cell, and LC3-II is also 48
Hour can be detected；And in SW480 cell, for c-PARP at 24 hours just with expression, LC3-II still can quilt at 48 hours
It detects (see Fig. 5 c and d).It is contaminated altogether in conjunction with above-mentioned Hoechst33258 and AO as a result, prompt us, the apoptosis of adenosine induction
Formerly, autophagy occurs rear.
(5) adenosine and autophagy inhibitor are combined effect；
Autophagy inhibitor chloroquine (CQ) or 3-MA (3-MA) and the processing of adenosine drug combination are sought in CCK8 experiment
Colon cancer cell effect.The results show that after added with adenosine and autophagy inhibitor drug, compared with cell of the list added with adenosine, carefully
Born of the same parents' survival rate is remarkably decreased.Data result has statistical significance.(P < 0.001 * P < 0.05, * * P < 0.01, * * *) (see Fig. 6)
Western blot have detected adenosine and autophagy inhibitor coprocessing to cell death related protein (c-PARP) with
The influence of autophagy GAP-associated protein GAP (LC3-I/II) expression.The results show that chloroquine inhibits lysosome to form autophagy in conjunction with autophagosome
Lysosome leads to the increase of storing up of autophagosome, therefore LC3-II expression enhancing.3-MA inhibits PI3K III activity,
Inhibit early stage autophagy process, the expression of LC3-II weakens.This all illustrates that autophagy process is significantly pressed down under autophagy inhibitor effect
System.In addition, apoptosis protein c-PARP is obviously increased after adenosine and autophagy inhibitor synergy, illustrate that Apoptosis increases
By force (see Fig. 6).
Apoptosis test experience confirms that autophagy inhibitor and adenosine are jointly processed by cell, increases the ratio of apoptotic cell.Chlorine
Quinoline and being jointly processed by for adenosine make SW620 apoptosis ratio increase to 29.3 ± 0.8%, SW480 Apoptosis from 15.6 ± 1.7%
Ratio increases to 70.2 ± 0.6% from 9.2 ± 0.3%.Data result has statistical significance.(P < 0.01 * *) similarly, 3-
Methyl adenine and adenosine are jointly processed by so that SW620 apoptosis ratio rises to 37.2 ± 0.3% from 15.6 ± 1.7%,
SW480 Apoptosis ratio increases to 72.3 ± 0.9% from 9.2 ± 0.3%.Data result have statistical significance (* * P <
0.01) (see Fig. 7).
In short, adenosine and autophagy inhibitor combination, clinically can be used as a kind of potential treatment colon cancer, reduce it is multiple
Hair and the high potency drugs of transfer.
The preferred embodiment of the present invention has been described in detail above, but the invention be not limited to it is described
Embodiment, those skilled in the art can also make various equivalent on the premise of not violating the inventive spirit of the present invention
Variation or replacement, these equivalent variation or replacement are all included in the scope defined by the claims of the present application.
Claims (10)
1. adenosine and autophagy inhibitor combination are preparing the application in treatment of colon cancer drug.
2. adenosine according to claim 1 and autophagy inhibitor combination are preparing the application in treatment of colon cancer drug,
It is characterized in that, the autophagy inhibitor is inhibition/downward autophagy related gene expression substance.
3. adenosine according to claim 2 and autophagy inhibitor combination are preparing the application in treatment of colon cancer drug,
It is characterized in that, the autophagy related gene is Beclin1 or ATG5.
4. adenosine according to claim 1 and autophagy inhibitor combination are preparing the application in treatment of colon cancer drug,
It is characterized in that, the autophagy inhibitor is the substance for inhibiting the protein expression of autophagy related gene Beclin1 or ATG5.
5. adenosine according to claim 1 and autophagy inhibitor combination are preparing the application in treatment of colon cancer drug,
It is characterized in that, the autophagy inhibitor is chloroquine, 3-MA, Bafilomycin A1, hydroxychloroquine or wortmannin.
6. adenosine according to claim 1 and autophagy inhibitor combination are preparing the application in treatment of colon cancer drug,
It is characterized in that, the autophagy inhibitor promotes Apoptosis of Colon Cancer Cells caused by adenosine.
7. a kind of pharmaceutical composition for treating colon cancer, which is characterized in that the pharmaceutical composition includes adenosine and autophagy suppression
Preparation and other pharmaceutically acceptable auxiliary material or carriers.
8. autophagy inhibitor is preparing the application in Apoptosis of Colon Cancer Cells promotor caused by adenosine.
9. autophagy inhibitor according to claim 8 is preparing answering in Apoptosis of Colon Cancer Cells promotor caused by adenosine
With, which is characterized in that the autophagy inhibitor is inhibition/downward autophagy related gene expression substance.
10. autophagy inhibitor according to claim 8 is in preparing Apoptosis of Colon Cancer Cells promotor caused by adenosine
Using, which is characterized in that the autophagy related gene is Beclin1 or ATG5.
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|---|---|---|---|---|
| CN114014901A (en) * | 2021-12-07 | 2022-02-08 | 天津科技大学 | Novel halogenated adenosine analogue 5' -bromodeoxyadenosine and preparation method and application thereof |
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| WO2006078774A2 (en) * | 2005-01-19 | 2006-07-27 | The Trustees Of The University Of Pennsylvania | Regulation of autophagy and cell survival |
| WO2012125486A1 (en) * | 2011-03-11 | 2012-09-20 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Combination chemotherapy for treating cancer |
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2018
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| WO2006078774A2 (en) * | 2005-01-19 | 2006-07-27 | The Trustees Of The University Of Pennsylvania | Regulation of autophagy and cell survival |
| WO2012125486A1 (en) * | 2011-03-11 | 2012-09-20 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Combination chemotherapy for treating cancer |
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